Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution.

The gut plays a key role in regulating metabolic health. Dietary factors disrupt intestinal physiology and contribute to obesity and diabetes, whereas bariatric procedures such as vertical sleeve gastrectomy (VSG) cause gut adaptations that induce robust metabolic improvements. However, our understanding of these adaptations at the cellular and molecular levels remains limited. In a validated murine model, we leverage single-cell transcriptomics to determine how VSG impacts different cell lineages of the small intestinal epithelium. We define cell type-specific genes and pathways that VSG rescues from high-fat diet perturbation and characterize additional rescue-independent changes brought about by VSG. We show that Paneth cells have increased expression of the gut peptide Reg3g after VSG. We also find that VSG restores pathways pertaining to mitochondrial respiration and cellular metabolism, especially within crypt-based cells. Overall, our study provides unprecedented molecular resolution of VSG's therapeutic effects on the gut epithelium.

A guide to establishing, implementing, and optimizing diversity, equity, inclusion, and accessibility (DEIA) committees.

Diversity, equity, inclusion, and accessibility (DEIA) efforts are increasingly recognized as critical for the success of academic institutions. These efforts are facilitated mainly through the formation of dedicated DEIA committees. DEIA committees enhance professional development and create a more inclusive environment, which benefits all members of the institution. Although leadership and faculty membership have recognized the importance and necessity of DEIA, the roles of DEIA committees may be more ambiguous. Although leadership and faculty may seek to support DEIA at their institutions, they may not always fully understand the necessity of these committees or how to successfully create a committee, foster and promote its success, and sustain its impact. Thus, here, we offer a background rationale and guide for strategically setting up DEIA committees for success and impact within an academic institution with applicability to scientific societies.

The Ldb1 transcriptional co-regulator is required for establishment and maintenance of the pancreatic endocrine lineage.

Pancreatic islet cell development is regulated by transcription factors (TFs) that mediate embryonic progenitor differentiation toward mature endocrine cells. Prior studies from our lab and others showed that the islet-enriched TF, Islet-1 (Isl1), interacts with the broadly-expressed transcriptional co-regulator, Ldb1, to regulate islet cell maturation and postnhyperatal function (by embryonic day (E)18.5). However, Ldb1 is expressed in the developing pancreas prior to Isl1 expression, notably in multipotent progenitor cells (MPCs) marked by Pdx1 and endocrine progenitors (EPs) expressing Neurogenin-3 (Ngn3). MPCs give rise to the endocrine and exocrine pancreas, while Ngn3+ EPs specify pancreatic islet endocrine cells. We hypothesized that Ldb1 is required for progenitor identity in MPC and EP populations during development to impact islet appearance and function. To test this, we generated a whole-pancreas Ldb1 knockout, termed Ldb1ΔPanc , and observed severe developmental and postnatal pancreas defects including disorganized progenitor pools, a significant reduction of Ngn3-expressing EPs, Pdx1HI ß-cells, and early hormone+ cells. Ldb1ΔPanc neonates presented with severe hyperglycemia, hypoinsulinemia, and drastically reduced hormone expression in islets, yet no change in total pancreas mass. This supports the endocrine-specific actions of Ldb1. Considering this, we also developed an endocrine-enriched model of Ldb1 loss, termed Ldb1ΔEndo . We observed similar dysglycemia in this model, as well as a loss of islet identity markers. Through in vitro and in vivo chromatin immunoprecipitation experiments, we found that Ldb1 occupies key Pdx1 and Ngn3 promoter domains. Our findings provide insight into novel regulation of endocrine cell differentiation that may be vital toward improving cell-based diabetes therapies.

Gut Factors Mediating the Physiological Impact of Bariatric Surgery.

Despite decades of obesity research and various public health initiatives, obesity remains a major public health concern. Our most drastic but most effective treatment of obesity is bariatric surgery with weight loss and improvements in co-morbidities, including resolution of type 2 diabetes (T2D). However, the mechanisms by which surgery elicits metabolic benefits are still not well understood. One proposed mechanism is through signals generated by the intestine (nutrients, neuronal, and/or endocrine) that communicate nutrient status to the brain. In this review, we discuss the contributions of gut-brain communication to the physiological regulation of body weight and its impact on the success of bariatric surgery. Advancing our understanding of the mechanisms that drive bariatric surgery-induced metabolic benefits will ultimately lead to the identification of novel, less invasive strategies to treat obesity.

LIM-domain transcription complexes interact with ring-finger ubiquitin ligases and thereby impact islet ß-cell function.

Diabetes is characterized by a loss of ß-cell mass, and a greater understanding of the transcriptional mechanisms governing ß-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA-binding protein 3 (SSBP3) regulates the genes necessary for ß-cell function, but few proteins are known to interact with this complex in ß-cells. To identify additional components, here we performed SSBP3 reverse-cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse ß-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for ß-cell function. We observed that Rnf20 and Rnf40 depletion reduces ß-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate ß-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.

The islet-expressed Lhx1 transcription factor interacts with Islet-1 and contributes to glucose homeostasis.

The LIM-homeodomain (LIM-HD) transcription factor Islet-1 (Isl1) interacts with the LIM domain-binding protein 1 (Ldb1) coregulator to control expression of key pancreatic ß-cell genes. However, Ldb1 also has Isl1-independent effects, supporting that another LIM-HD factor interacts with Ldb1 to impact ß-cell development and/or function. LIM homeobox 1 (Lhx1) is an Isl1-related LIM-HD transcription factor that appears to be expressed in the developing mouse pancreas and in adult islets. However, roles for this factor in the pancreas are unknown. This study aimed to determine Lhx1 interactions and elucidate gene regulatory and physiological roles in the pancreas. Co-immunoprecipitation using ß-cell extracts demonstrated an interaction between Lhx1 and Isl1, and thus we hypothesized that Lhx1 and Isl1 regulate similar target genes. To test this, we employed siRNA-mediated Lhx1 knockdown in ß-cell lines and discovered reduced Glp1R mRNA. Chromatin immunoprecipitation revealed Lhx1 occupancy at a domain also known to be occupied by Isl1 and Ldb1. Through development of a pancreas-wide knockout mouse model ( Lhx1∆Panc), we demonstrate that aged Lhx1∆Panc mice have elevated fasting blood glucose levels, altered intraperitoneal and oral glucose tolerance, and significantly upregulated glucagon, somatostatin, pancreatic polypeptide, MafB, and Arx islet mRNAs. Additionally, Lhx1∆Panc mice exhibit significantly reduced Glp1R, an mRNA encoding the insulinotropic receptor for glucagon-like peptide 1 along with a concomitant dampened Glp1 response and mild glucose intolerance in mice challenged with oral glucose. These data are the first to reveal that the Lhx1 transcription factor contributes to normal glucose homeostasis and Glp1 responses.

Age-dependent changes cooperatively impact skeletal muscle regeneration after compartment syndrome injury.

Declining skeletal muscle function, due to injury and aging (sarcopenia), results in a significantly decreased quality of life and is a major cause of disability in the United States. Studies examining recovery from muscle injury in models of older animals principally used insults that primarily affect only the myofibers without affecting the muscle tissue microenvironment. This type of injury does not adequately represent the full extent of tissue damage observed in older humans, which encompasses injury not only to the muscle fibers, but also to the surrounding tissue components, such as the vasculature and nerves. Previously, we described a novel rat model of compression-induced muscle injury that results in multicomponent injury to the muscle and adequately mimics compartment syndrome injuries seen in patients. Herein, we characterized tissue regeneration in young, adult, and aged rats after compartment syndrome injury. We observed significant differences between the regeneration process in the different aged rats that involved muscle function, tissue anatomical features, neovascularization, and innervation. Compared to young rats, adult rats had delayed functional recovery, whereas the aged rats were deficient in their regenerative capacity. Age-dependent changes in both the ability to restore the contractile apparatus and myogenesis are important, and must be taken into consideration when designing therapies for the treatment of muscle injury.

The SSBP3 co-regulator is required for glucose homeostasis, pancreatic islet architecture, and beta-cell identity.

OBJECTIVE: Transcriptional complex activity drives the development and function of pancreatic islet cells to allow for proper glucose regulation. Prior studies from our lab and others highlighted that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), and its interacting co-regulator, Ldb1, are vital effectors of developing and adult ß-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with Isl1 and Ldb1 in ß-cells and primary islets (mouse and human) to impact ß-cell target genes MafA and Glp1R in vitro. Members of the SSBP family stabilize TF complexes by binding directly to Ldb1 and protecting the complex from ubiquitin-mediated turnover. In this study, we hypothesized that SSBP3 has critical roles in pancreatic islet cell function in vivo, similar to the Isl1::Ldb1 complex. METHODS: We first developed a novel SSBP3 LoxP allele mouse line, where Cre-mediated recombination imparts a predicted early protein termination. We bred this mouse with constitutive Cre lines (Pdx1- and Pax6-driven) to recombine SSBP3 in the developing pancreas and islet (SSBP3ΔPanc and SSBP3ΔIslet), respectively. We assessed glucose tolerance and used immunofluorescence to detect changes in islet cell abundance and markers of ß-cell identity and function. Using an inducible Cre system, we also deleted SSBP3 in the adult ß-cell, a model termed SSBP3Δß-cell. We measured glucose tolerance as well as glucose-stimulated insulin secretion (GSIS), both in vivo and in isolated islets in vitro. Using islets from control and SSBP3Δß-cell we conducted RNA-Seq and compared our results to published datasets for similar ß-cell specific Ldb1 and Isl1 knockouts to identify commonly regulated target genes. RESULTS: SSBP3ΔPanc and SSBP3ΔIslet neonates present with hyperglycemia. SSBP3ΔIslet mice are glucose intolerant by P21 and exhibit a reduction of ß-cell maturity markers MafA, Pdx1, and UCN3. We observe disruptions in islet cell architecture with an increase in glucagon+ α-cells and ghrelin+ ε-cells at P10. Inducible loss of ß-cell SSBP3 in SSBP3Δß-cell causes hyperglycemia, glucose intolerance, and reduced GSIS. Transcriptomic analysis of 14-week-old SSBP3Δß-cell islets revealed a decrease in ß-cell function gene expression (Ins, MafA, Ucn3), increased stress and dedifferentiation markers (Neurogenin-3, Aldh1a3, Gastrin), and shared differentially expressed genes between SSBP3, Ldb1, and Isl1 in adult ß-cells. CONCLUSIONS: SSBP3 drives proper islet identity and function, where its loss causes altered islet-cell abundance and glucose homeostasis. ß-Cell SSBP3 is required for GSIS and glucose homeostasis, at least partially through shared regulation of Ldb1 and Isl1 target genes.

ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Preproglucagon Products and Their Respective Roles Regulating Insulin Secretion.

Historically, intracellular function and metabolic adaptation within the α-cell has been understudied, with most of the attention being placed on the insulin-producing ß-cells due to their role in the pathophysiology of type 2 diabetes mellitus. However, there is a growing interest in understanding the function of other endocrine cell types within the islet and their paracrine role in regulating insulin secretion. For example, there is greater appreciation for α-cell products and their contributions to overall glucose homeostasis. Several recent studies have addressed a paracrine role for α-cell-derived glucagon-like peptide-1 (GLP-1) in regulating glucose homeostasis and responses to metabolic stress. Further, other studies have demonstrated the ability of glucagon to impact insulin secretion by acting through the GLP-1 receptor. These studies challenge the central dogma surrounding α-cell biology describing glucagon's primary role in glucose counterregulation to one where glucagon is critical in regulating both hyper- and hypoglycemic responses. Herein, this review will update the current understanding of the role of glucagon and α-cell-derived GLP-1, placing emphasis on their roles in regulating glucose homeostasis, insulin secretion, and ß-cell mass.

Prolactin Receptor Signaling Regulates a Pregnancy-Specific Transcriptional Program in Mouse Islets.

Pancreatic ß-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram ß-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes mellitus (GDM). We recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where ß-cell-specific PRLR knockout (ßPRLRKO) mice exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within ß-cells during pregnancy, we performed a transcriptomic analysis of pancreatic islets isolated from either ßPRLRKO mice or littermate controls in late gestation. Gene set enrichment analysis identified forkhead box protein M1 and polycomb repressor complex 2 subunits, Suz12 and enhancer of zeste homolog 2 (Ezh2), as novel candidate regulators of PRLR-dependent ß-cell adaptation. Gene ontology term pathway enrichment revealed both established and novel PRLR signaling target genes that together promote a state of increased cellular metabolism and/or proliferation. In contrast to the requirement for ß-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat diet feeding. Collectively, the current study expands our understanding of which transcriptional regulators and networks mediate gene expression required for islet adaptation during pregnancy. The current work also supports the presence of pregnancy-specific adaptive mechanisms distinct from those activated by nutritional stress.

* The Impact of Age on Skeletal Muscle Progenitor Cell Survival and Fate After Injury.

Sarcopenia is defined as the loss of skeletal muscle mass and function due to age, and represents a major cause of disability in the elderly population. The contributing factors to the onset of sarcopenia are not well defined, but appear to involve age-dependent changes in both the tissue microenvironment and muscle progenitor cell (MPC) population. MPC transplantation has the potential to be a novel therapy for treatment of muscle dysfunction due to aging or injury, but has not shown significant clinical efficacy to date. The goal of this research was to use a rat model of skeletal muscle injury to examine the differential effects of age on MPC survival, differentiation, and tissue regeneration after transplantation. Fluorescently labeled MPCs, derived from young (YMPCs) and adult (AMPCs) donor rats, were transplanted in the injured tibialis anterior (TA) muscles of young, adult, and aged rats. Our results demonstrated that integration and maturation of YMPCs into mature myofibers were dependent on the age of the host microenvironment; whereas, the integration and maturation of AMPCs were less dependent on age and more dependent on intrinsic cellular changes. These data suggest that the age of both the host microenvironment and cells for transplantation must be considered when designing cell therapy regimens.

Islet encapsulation with polyphenol coatings decreases pro-inflammatory chemokine synthesis and T cell trafficking.

Type 1 Diabetes (T1D) is a chronic pro-inflammatory autoimmune disease consisting of islet-infiltrating leukocytes involved in pancreatic ß-cell lysis. One promising treatment for T1D is islet transplantation; however, clinical application is constrained due to limited islet availability, adverse effects of immunosuppressants, and declining graft survival. Islet encapsulation may provide an immunoprotective barrier to preserve islet function and prevent immune-mediated rejection after transplantation. We previously demonstrated that a novel cytoprotective nanothin multilayer coating for islet encapsulation consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), was efficacious in dampening in vitro immune responses involved in transplant rejection and preserving in vitro islet function. However, the ability of (PVPON/TA) to maintain islet function in vivo and reverse diabetes has not been tested. Recent evidence has demonstrated that modulation of redox status can affect pro-inflammatory immune responses. Therefore, we hypothesized that transplanted (PVPON/TA)-encapsulated islets can restore euglycemia to diabetic mice and provide an immunoprotective barrier. Our results demonstrate that (PVPON/TA) nanothin coatings can significantly decrease in vitro chemokine synthesis and diabetogenic T cell migration. Importantly, (PVPON/TA)-encapsulated islets restored euglycemia after transplantation into diabetic mice. Our results demonstrate that (PVPON/TA)-encapsulated islets may suppress immune responses and enhance islet allograft acceptance in patients with T1D.

LDB1 Regulates Energy Homeostasis During Diet-Induced Obesity.

The broadly expressed transcriptional coregulator LDB1 is essential for ß-cell development and glucose homeostasis. However, it is unclear whether LDB1 has metabolic roles beyond the ß-cell, especially under metabolic stress. Global Ldb1 deletion results in early embryonic lethality; thus, we used global heterozygous Ldb1+/- and inducible ß-cell-specific Ldb1-deficient (Ldb1Δß-cell) mice. We assessed glucose and insulin tolerance, body composition, feeding, and energy expenditure during high-fat diet exposure. Brown adipose tissue (BAT) biology was evaluated by thermogenic gene expression and LDB1 chromatin immunoprecipitation analysis. We found that partial loss of Ldb1 does not impair the maintenance of glucose homeostasis; rather, we observed improved insulin sensitivity in these mice. Partial loss of Ldb1 also uncovered defects in energy expenditure in lean and diet-induced obese (DIO) mice. This decreased energy expenditure during DIO was associated with significantly altered BAT gene expression, specifically Cidea, Elovl3, Cox7a1, and Dio2. Remarkably, the observed changes in energy balance during DIO were absent in Ldb1Δß-cell mice, despite a similar reduction in plasma insulin, suggesting a role for LDB1 in BAT. Indeed, LDB1 is expressed in brown adipocytes and occupies a regulatory domain of Elovl3, a gene crucial to normal BAT function. We conclude that LDB1 regulates energy homeostasis, in part through transcriptional modulation of critical regulators in BAT function.

SSBP3 Interacts With Islet-1 and Ldb1 to Impact Pancreatic ß-Cell Target Genes.

Islet-1 (Isl1) is a Lin11, Isl1, Mec3 (LIM)-homeodomain transcription factor important for pancreatic islet cell development, maturation, and function, which largely requires interaction with the LIM domain-binding protein 1 (Ldb1) coregulator. In other tissues, Ldb1 and Isl1 interact with additional factors to mediate target gene transcription, yet few protein partners are known in ß-cells. Therefore, we hypothesize that Ldb1 and Isl1 participate in larger regulatory complexes to impact ß-cell gene expression. To test this, we used cross-linked immunoprecipitation and mass spectrometry to identify interacting proteins from mouse ß-cells. Proteomic datasets revealed numerous interacting candidates, including a member of the single-stranded DNA-binding protein (SSBP) coregulator family, SSBP3. SSBPs potentiate LIM transcription factor complex activity and stability in other tissues. However, nothing was known of SSBP3 interaction, expression, or activity in ß-cells. Our analyses confirmed that SSBP3 interacts with Ldb1 and Isl1 in ß-cell lines and in mouse and human islets and demonstrated SSBP3 coexpression with Ldb1 and Isl1 pancreas tissue. Furthermore, ß-cell line SSBP3 knockdown imparted mRNA deficiencies similar to those observed upon Ldb1 reduction in vitro or in vivo. This appears to be (at least) due to SSBP3 occupancy of known Ldb1-Isl1 target promoters, including MafA and Glp1r. This study collectively demonstrates that SSBP3 is a critical component of Ldb1-Isl1 regulatory complexes, required for expression of critical ß-cell target genes.